PROJECT SUMMARY/ABSTRACT Cell-cell interactions are involved in numerous biological processes, including immune responses, embryonic development, neuronal signaling and cancer metastasis. Some major gaps in our understanding of cell-cell interactions include: what are the interacting partners of a specific cell type at different stages of a biological process? How to modify the cell-surface landscape of these interacting partners to tune the functions of a specific cell type with minimum perturbation of its endogenous functions? The objective of this project is to develop glycosyltransferase-based chemoenzymatic methods to incorporate biopolymers, including enzymes and monoclonal antibodies, onto the cell surface to study these puzzles. In Aim 1 of this project, we will identify or engineer a promiscuous glycosyltransferase that can transfer biopolymer-functionalized unnatural sugars from the corresponding nucleotide sugar donors to the cell-surface glycocalyx of the cells of interest. When the bioploymer is an enzyme, it will enable the proximity-dependent labeling of prey cells that interact with the cells of interest (bait cells) that harbor the enzyme, providing that the cell surface of prey cells expresses the substrates to be modified by the enzyme. Subsequently, the signal generated by the labeling can be detected and analyzed ex vivo by flow cytometry. In Aim 1d and Aim 2, we will apply this method to probe cell-cell interactions in vivo using mouse models. The focus will be the identification of interacting partners of specific immune cell types during immune activation and T-cell exhaustion (T-cell exhaustion is the process during which T cells progressively lose their functions). Finally, we will develop a method in Aim 3 to conjugate monoclonal antibodies onto immune cells to endow them with specific targeting capabilities. The migration and anti-tumor efficacy of the modified cells will be evaluated in mouse tumor models.